Hi Sanchun,

We commit a tissue culture sin when rescuing influenza viruses. We make a co-culture of 293T (8x10^5 cells/well) and MDCK (3x10^5 cells/well). 293T cells alone are great for transfection, but they're very poor at growing flu. Adding the MDCK cells provides a good matrix for the 293T cells, and a good substrate for flu once it starts to replicate. We transfect for 6 hours in Opti-MEM, and then replace the media. At 24 hours post transfection, we add fresh Opti-MEM supplemented with 1µg/mL TPCK-treated Trypsin. The trypsin is required to cleave the HA protein into its mature form. If your 293T cells are making it as long as you say, you're not using trypsin!

Following the addition of trypsin, we incubate the cells for 2-3 additional days. The 293T cells will start to look bad because of the trypsin, but the MDCK cells will continue to grow. Once the virus launches, you should start to see CPE in the MDCK cells. If you don't see anything, do a blind passage onto MDCK cells in the presence of TPCK-trypsin.

PR8 should rescue very well. Where did you get the PR8 plasmids? Are you sure that they're bidirectional vectors (pHW200 or pDZ)? If not, you may need to obtain "helper" plasmids for PB2, PB1, PA, and NP that express these proteins from an RNA polymerase II promoter.

As long as we are depended on hen eggs for research, rather than mammalian cell lines, we are going to be hampered by time delays, and miscues.

Hi Sanchun, what volume are you using to transfect the plasmids in? Do you use a T-25 flask or plates for transfections? Do you use helper plasmids just to give the replicase a boost?

Have you tried FUGENE as transfectant? Also, a co-culture with MDCK can help to rescue the virus (without a CPE being observed).

Hi,

we transfect with the old and cheap Cacluim-phosphate technique, which should give about 80% transfection efficiency (or more) - so you have a good chance to get al eight plasmids into one cell. We always seed a 1:1 mixture of 293T and MDCK cells, as MDCK cells produce more virus. Did you add trypsin, which is needed for propagation of PR8 (and for most strains) - we add ist one or two days after transfection.

Good luck,

Michael

I have not handled influenza. Yet, as I see it influenza should not result in syncytia formation. Heavily infected cells might come afloat but it will be difficult to visualize if you plate 100 infectious viruses in a plate. Protocols for influenza are established and you should scan through them. I am inclined to think that the virus may have problems even when it comes out of the infected/transfected cell in soft agar and one may have to wait for longer periods of time than in case of paramyxoviruses like measles. As it is co-transfection with 8 segments corresponding to antigenome/genome will have lower probability of success than single pieces. I would also like to transfer the supernatant a few cycles and view the infected cells under EM for assembly of any viruses.

Hi Sanchun, you can discard your sample. If you don't have a good CPE after 3-4 days, the experiment failed. 200µl supernatant from 293T sounds quite small anyways.

We use LF2000 since ~10 years now for rescuing influenza of many different subtypes and species, and it works almost every time. But you can try other transfection reagent/methods as well to find which suits you best.

Questions to help you further would be: what volume/format are you using for rescue (i.e 6well Plates or Flasks?) How much DNA did you transfect? Did you try to infect eggs with your 293T supernatant in parallel?

The HA of PR8 has a monobasic cleavage site. Which amount of trypsin did you add to activate the HA?

Rescue efficiency of PR8 plasmids is quite high. From your post, I think your rescue attempt failed this time. It would also help if you perform HA test to where you inoculated your supernatants (e.g., MDCK cells). As mentioned by above comments, you have to eliminate one by one the potential source of error. First is the type of vessel (T-flask, 6-well, or 12-well) that you used. Be sure to use appropriate ratios of DNA to transfection reagent and replace transfection media at least 4-6 hrs and replace with a reduced serum media. Reconsider your cells (293T alone or a co-culture of 293T and MDCK) too or the use of helper plasmids, and of course the addition of trypsin at least 24 (or 36) hrs post-transfection. There is also a little trick with the trypsin since 293T easily detaches from the vessel. You can start using low concentrations first then gradually increase as you advance depending on whether your 293T cells are still confluent. Lastly, you can also use eggs in parallel to the MDCK cells to rescue your newly reconstituted virus. Goodluck!

Hi Sanchun,

Unfortunately, it seems that your experiment has failed. We use TransIT reagent from Mirus for transfection and it works well for us. Also, make sure that your 293T cells are not grown to confluence for transfection. If transfection was successful, you should be able to visualize CPE on MDCK cells within 2 days.

Good luck!

A

Hi Sanchun,

We commit a tissue culture sin when rescuing influenza viruses. We make a co-culture of 293T (8x10^5 cells/well) and MDCK (3x10^5 cells/well). 293T cells alone are great for transfection, but they're very poor at growing flu. Adding the MDCK cells provides a good matrix for the 293T cells, and a good substrate for flu once it starts to replicate. We transfect for 6 hours in Opti-MEM, and then replace the media. At 24 hours post transfection, we add fresh Opti-MEM supplemented with 1µg/mL TPCK-treated Trypsin. The trypsin is required to cleave the HA protein into its mature form. If your 293T cells are making it as long as you say, you're not using trypsin!

Following the addition of trypsin, we incubate the cells for 2-3 additional days. The 293T cells will start to look bad because of the trypsin, but the MDCK cells will continue to grow. Once the virus launches, you should start to see CPE in the MDCK cells. If you don't see anything, do a blind passage onto MDCK cells in the presence of TPCK-trypsin.

PR8 should rescue very well. Where did you get the PR8 plasmids? Are you sure that they're bidirectional vectors (pHW200 or pDZ)? If not, you may need to obtain "helper" plasmids for PB2, PB1, PA, and NP that express these proteins from an RNA polymerase II promoter.

Hi, it sounds like the approach failed.

I would use TransLIT ins tread of Lipofectamine. Have you used TPCK-Trypsin in the cell media?

Eike

PR8 should be easy to reascue. One problem might be the transfection reagent , make sure you do a control GFP reporter transfection.

To confirm efficient transfection I have also used one of Ron Fouchier's minireplicon reporter plasmids that only expresses GFP when all the influenza polymerase proteins are expressed in the cell (http://www.ncbi.nlm.nih.gov/pubmed/17374773). That lets you know at least four of the plasmids have got into an individual 293T cell. To increase the number of plasmids getting into a cell I have also used Vincent Bourret's ENV plasmid which causes the cells to join and form karyons (http://www.ncbi.nlm.nih.gov/pubmed/23009349).

The suggestions from Matthew Angel and Philippe Noriel Pascua are very useful. I use a 20:1 ratio of 293T:MDCK cells.

The previous responses cover most of the topics to address but I would like to add a couple fo things. The brand of transfection reagent shouldn't matter unless it is expired. I've rescued PR8 8-plasmid with pretty much every reagent available, 293T alone or mixed culture. One response suggested a GFP reporter control, which is fine, but our lab actually uses PR8 rescue as a control for other transfections because it is so reliable. Keep your cell confluence under 100%, wash serum away and use serum free media during the transfection, add TPCK-Trypsin at 48-72 hour to cleave the virus. If you are using the pHW19x plasmids from St Jude/Medimmune, shoot me an e-mail and I can send you our protocal. You may also run a gel on the plasmid preps to verify they are running properly. the PB2 plasmid has a tendancy to go abhorrent after many freeze-thaw cycles or improper storage, losing supercoiled and forming a high species concatemer?.

With the first passage, if you don't see CPE or get RBC agglutination after a couple of days, it simply didn't work and waiting longer won't help.

Everybody rescues infectious viruses after cDNA transfection, except me. So depressed!

From my experience, you should maintain the cells transfected with the plasmids for up to one week. After that, you can collect the supernatants, and infect MDCK cells with them. In general, the 293 or 293T cells transfected with the plasmids does not show apparent CPE, because of the limited amount of the viruses that were generated by the plasmid-based reverse genetics.

I am wondering if you transfected the 293 or 293 cells with 8 plasmids (pPol) that express the viral genes (PB2, PB1, PA, HA, NP, NA, M, and NS genes) together with 4 plasmids (pCAGGS) that express the viral polymerase complex proteins (PB2, PB1, PA, and NP).

I think most of the details have been covered from previous comments. Anyway, if you still need a protocol, do not hesitate to ask. BTW, as Ryo Takano said, which can of plasmids do you use? Are they bidirectional?

What RNA polymerse did you used in reverse genetic system?

the T7 polymerase will transcribe a 5' tri-p-RNA, which induced the RIG-I pathway and killed the few rescued virus. I think this is the reason that poor/no titer you can get from 293T. if you want to use 293T cells alone, I think you can cotransfect pCAGGS-NS1 to block the immune response.

Hi Shanchun Wu,

A complete set of eight dual promoter plasmids (1 µg each) of PR8

was co-transfected into a co-culture of 293T/MDCK-II cells (ratio

3:1) in Opti-MEM (5%FCS and no Pen/Strep). Briefly,

the transfection mixture consisting of 180 µl “serum free and antibiotic free Opti-MEM” and 8 µg

of plasmid DNA (1 µg of each plasmid) along with 16-18 µl “Trans-

IT2020” was incubated for 45 min at room temperature. Then, the

mixture was diluted to 1 ml using “Opti-MEM” and transferred to a

nearly confluent cell monolayer. The cells were then incubated for

12 h at 37 ◦C in the presence of 5% CO2. On the next day, the transfection

media were replaced with 1 ml of “Opti-MEM” containing

penicillin/streptomycin and 0.2% bovine serum albumin and the

cell cultures were incubated for 24 h. On the next day, an additional

1 ml of “Opti-MEM” containing P/S, 0.2% BSA and 2 µg/ml TPCKtreated

trypsin (Sigma–Aldrich, USA) was added to proteolytically

cleave the hemagglutinin (HA) and thus activate its fusion activity.

The cell culture supernatant was harvested 48–72 h after addition

of the TPCK-treated trypsin and cell debris was removed by centrifugation

at 1500 rpm for 5 min at 4 ◦C. A 500 µl aliquot of each

supernatant was used to inoculate MDCK-II cells, which were then

incubated for 72 h in the presence of 1 µg/ml TPCK-treated trypsin.

The viruses rescued from these cells were stored at −80 ◦C until use.